An example of the use of fluorescence detection is in nucleic acid testing (NAT). This is a core element in molecular diagnostics for detecting genetic predispositions for diseases, for determining RNA expression levels or identification of pathogens, like bacteria and viruses that cause infections.
In many cases, particularly in the identification of pathogens, the amount of target DNA present in a reasonable sample volume is very low, and this does not allow direct detection. Amplification techniques are necessary to obtain detectable quantities of the target material. Different amplification techniques have been proposed and are used in daily practice. The most widely used are based on the so-called Polymerase chain reaction (PCR).
The amplification involves the denaturing of double-stranded DNA at elevated temperature (typically >90 degrees Celsius), specific binding of primers to the DNA sample at a reduced temperature (approximately 65 degrees) and copying of the original sequences starting from the primer position (at approximately 70 degrees). This procedure is repeated and in every cycle the amount of DNA with the specific sequence is doubled (when proceeding at 100% efficiency).
After amplification, the presence of target DNA is detected by measuring the fluorescence intensity of the labeled amplified DNA, for instance after electrophoretic separation in a capillary or after hybridization to so-called capture probes which are applied in spots on a surface over which the amplification product is flowed.
This invention relates to the apparatus used to provide the illumination to the sample, and the method of use.
The standard technique for fluorescence detection is the use of a scanning confocal microscope. Typically, a small (<1 μm), diffraction limited spot is used to excite the fluorescence in the focal plane. In the detection part of the system, only the light resulting from this single excitation point is detected.
It has previously been proposed that the excitation of a number of spots or a complete line in parallel enables an increase in the scanning speed, without a major impact on the confocality of the detection system. A pixilated detector can be used to detect the fluorescent emission.
The main disadvantage of a camera is the readout speed and the fact that these detectors are rather bulky and expensive. It is the aim of this invention to overcome these problems by sacrificing the spatial resolution along the length of the excitation line. A more compact solution is important for Point of Care test solutions.